The New World clade B arenaviruses (NWA), particularly Junin (JUNV) and Machupo (MACV) are associated with outbreaks of viral hemorrhagic fever in South America. JUNV and MACV infections represent zoonoses from rodents and are both transmitted through aerosols; thus, the NWAs are potential bioterrorism agents. JUNV in particular is one of only three viruses included in the recent list of agents developed by the Department of Homeland Security that represent in their assessment the most significant bioterrorism threats faced by the US. Recently, transferrin receptor 1 (TfR1) was identified as a cellular receptor for the pathogenic New World arenaviruses. However, little is known about the steps subsequent to virus/TfR1 cell surface interaction and there is evidence that other molecules play a role in NWA entry. Moreover, although JUNV and MACV efficiently use TfR1 from their host rodent species for entry, they appear to only be pathogenic in infected humans. We propose several approaches to identify co-factors that support NWA infection as a means for better understanding the pathogenicity of these viruses in humans. These include cDNA expression, siRNA and chemical library screening, to identify genes and pathways that could serve as targets for therapeutic intervention of infection. All rely on the high-throughput, cell-based screening (HTCS) core at Penn, that will support projects in RCE Programs I and II. We also propose creating chimeric JUNV/MMTV hybrid viruses using the MMTV envelope and JUNV wild type and vaccine strain (candid #1) GP proteins to pseudotype JUNV and MMTV virus cores, respectively. These pseudotypes will provide us with a means of studying how arenavirus gene products cause pathogenicity. The chimeric viruses will be used for HTCS screens of NFicB and IRF3 reporter cells, to begin to uncover the activation pathways that lead to the cytokine storm. As there are currently no anti-virals in use for therapeutic or postinfection prophylactic treatment of the clade B arenaviruses, the identification of co-factors that participate in JUNV and MACV entry and perhaps contribute to pathogenesis has the potential to lead to the development of novel therapies for hemorrhagic fevers cause by these viruses.